Bi-allelic loss-of-function variants in PPFIBP1 cause a neurodevelopmental disorder with microcephaly, epilepsy, and periventricular calcifications.

PPFIBP1 encodes for the liprin-ß1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.

A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy.

The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

Age-related changes in the inflammatory responses to viral infections in the central nervous system during childhood.

BACKGROUND: The developmental stages and function of immune cells in the central nervous system during infancy and childhood are poorly understood. We analyzed whether cytokine and chemokine profiles in children and adolescents with viral central nervous system infections were different depending on age. METHODS: The acute phase cerebrospinal fluid of 80 children (mean age 98 months, range 1-206 months) were analyzed for protein levels of interleukin-1ß (IL-1ß), IL-1-RA, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, monocyte chemoattractant protein-1 (MCP-1), interferon (IFN) gamma-induced protein 10 (IP-10), IFN-γ, and macrophage migration inhibitory factor (MIF). RESULTS: We found an age-dependent increased expression of IL-4, IL-6, IL-13, MIF, IP-10, and IFN-γ and a decreased expression of MCP-1 and IL-15 in response to a viral infection of the central nervous system. In contrast, all other cytokines and chemokine were unaffected by the age of the patient. CONCLUSION: These findings demonstrate that the immunological response to a viral infection matures during childhood and adolescence. This may in turn be of importance for the outcome of a viral infection and the risk for subsequent sequela. It also demonstrates that age is a factor that needs to be considered when using cytokines and chemokines as biomarkers for infections in the central nervous system. IMPACT: The immunological response to a viral infection matures during childhood and adolescence. This may be of importance for the outcome of a viral infection and the risk for subsequent sequela. It also demonstrates that age is a factor that needs to be considered when using cytokines and chemokines as biomarkers for infections in the central nervous system.

Clinical guidance for diagnosis and management of suspected Pediatric Acute-onset Neuropsychiatric Syndrome in the Nordic countries.

Pediatric acute-onset neuropsychiatric syndrome is a clinical concept used to describe a subgroup of children with sudden onset of psychiatric and somatic symptoms. The diagnostic term and especially management of children differs depending on the clinical setting to which they present, and the diagnosis and management is controversial. The aim of this paper is to propose a clinical guidance including homogenous diagnostic work-up and management of paediatric acute onset neuropsychiatric syndrome within the Nordic countries. The guidance is authored by a Nordic-UK working group consisting of paediatric neurologist, child psychiatrists and psychologists from Denmark, Norway, Sweden and Great Britain, and is the result of broad consensus. CONCLUSION: Consensus was achieved in the collaboration on work-up and treatment of patients with paediatric acute-onset neuropsychiatric syndrome, which we hope will improve and homogenise patient care and enable future collaborative research in the field.

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly.

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype-phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype-phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors.

Human In Vitro Models of Neuroenergetics and Neurometabolic Disturbances: Current Advances and Clinical Perspectives.

Neurological conditions conquer the world; they are the leading cause of disability and the second leading cause of death worldwide, and they appear all around the world in every age group, gender, nationality, and socioeconomic class. Despite the growing evidence of an immense impact of perturbations in neuroenergetics on overall brain function, only little is known about the underlying mechanisms. Especially human insights are sparse, owing to a shortage of physiologically relevant model systems. With this perspective, we aim to explore the key steps and considerations involved in developing an advanced human in vitro model for studying neuroenergetics. We discuss biological and technological strategies to meet the requirements of a predictive model, aiming at providing a guide and inspiration for future in vitro models of neuroenergetics.

Neurocognitive outcome in children and adolescents following infectious encephalitis.

Infectious encephalitis in children is fairly uncommon, but unfavorable outcomes are seen in many survivors. The aim of this study was to prospectively describe the long-term neurocognitive consequences following infectious encephalitis in childhood. Children admitted to a primary and tertiary hospital in Sweden between 2011 and 2016 were asked to participate. Fifty-nine children were assessed at a median time of 18 months (IQR 18-20) after hospitalization. Follow-up included measures of intellectual functioning, attention, working memory, and executive functions. Caregiver ratings of executive functioning and behavioral - emotional symptoms were assessed with standardized questionnaires. Neurocognitive outcome and measures of executive functions and behavioral-emotional symptoms varied greatly among participants. Basic auditory attention, working memory, and mental processing speed were affected and significantly lower compared to a standardized mean. Other domains identified as areas of vulnerability included executive functions, sustained attention, and the exert of self-control. Behavioral-emotional symptoms were less common; however, somatic complaints and behaviors related to conduct problems were seen in about one-third of individuals. This study highlights the importance of a comprehensive neurocognitive examination to identify children with unfavorable outcomes.

The Swedish COG6-CDG experience and a comprehensive literature review.

Here, we present the first two Swedish cases of Conserved Oligomeric Golgi complex subunit 6-congenital disorders of glycosylation (COG6-CDG). Their clinical symptoms include intellectual disability, Attention Deficit/Hyperactivity Disorder (ADHD), delayed brain myelinization, progressive microcephaly, joint laxity, hyperkeratosis, frequent infections, and enamel hypoplasia. In one family, compound heterozygous variants in COG6 were identified, where one (c.785A>G; p.Tyr262Cys) has previously been described in patients of Moroccan descent, whereas the other (c.238G>A; p.Glu80Lys) is undescribed. On the other hand, a previously undescribed homozygous duplication (c.1793_1795dup) was deemed the cause of the disease. To confirm the pathogenicity of the variants, we treated patient and control fibroblasts with the ER-Golgi transport inhibitor Brefeldin-A and show that patient cells manifest a significantly slower anterograde and retrograde ER-Golgi transport.

The developing immune system - from foetus to toddler.

UNLABELLED: During foetal development, neonatal period and childhood, the immune system is constantly maturing. In the foetus, infection responsiveness is low and associates with spontaneous abortion. During the neonatal period, the infection response shifts towards a more pro-inflammatory response. The immune system of the newborn acquires adaptive features as a result of exposure to microbes. CONCLUSION: The development of the human immune system is a continuous process where both accelerated and retarded development is deleterious.

Expanding the phenotype of the recurrent truncating eIF2γ pathogenic variant p.(Ile465Serfs*4) identified in two brothers with MEHMO syndrome.

We describe two brothers with a recurrent truncating EIF2S3 variant and MEHMO (Mental retardation, Epileptic seizures, Hypogonadism and -genitalism, Microcephaly, Obesity). Both had the previously described facial dysmorphic features, microcephaly, developmental impairment, hypoglycemia, hypothyreosis, diabetes mellitus, epilepsy, hypertonus, obesity, and micropenis. Additionally, we describe hypothermia and reduced umbilical blood flow.

Case report: Fatal outcome of pyridoxine-dependent epilepsy presenting as respiratory distress followed by a circulatory collapse.

Pyridoxine-dependent epilepsy is a rare autosomal recessive disease usually associated with neonatal seizures that do not respond to common antiseizure medications but are controlled by pyridoxine administration. Because the symptoms can mimic common neonatal disorders, the diagnosis can be initially missed or delayed. We report a fatal case of a boy who was initially diagnosed with respiratory distress, birth asphyxia, and persistent pulmonary hypertension and whose condition rapidly deteriorated during the first day of life.

A Missense Variant in PDK1 Associated with Severe Neurodevelopmental Delay and Epilepsy.

The pyruvate dehydrogenase complex (PDC) is responsible for the conversion of pyruvate into acetyl-CoA, which is used for energy conversion in cells. PDC activity is regulated by phosphorylation via kinases and phosphatases (PDK/PDP). Variants in all subunits of the PDC and in PDK3 have been reported, with varying phenotypes including lactic acidosis, neurodevelopmental delay, peripheral neuropathy, or seizures. Here, we report a de novo heterozygous missense variant in PDK1 (c.1139G > A; p.G380D) in a girl with developmental delay and early onset severe epilepsy. To investigate the role of PDK1G380D in energy metabolism and neuronal development, we used a zebrafish model. In zebrafish embryos we show a reduced number of cells with mitochondria with membrane potential, reduced movements, and a delay in neuronal development. Furthermore, we observe a reduction in the phosphorylation of PDH-E1α by PDKG380D, which suggests a disruption in the regulation of PDC activity. Finally, in patient fibroblasts, a mild reduction in the ratio of phosphorylated PDH over total PDH-E1α was detected. In summary, our findings support the notion that this aberrant PDK1 activity is the cause of clinical symptoms in the patient.

Altered cytokine levels in cerebrospinal fluid following ketogenic diet of children with refractory epilepsy.

Ketogenic diet is an effective treatment which has the potential to achieve a significant seizure reduction in drug-resistant epilepsy. The mechanism behind this effect is unclear, but one hypothesis is that the mechanism is anti-inflammatory. In this prospective study on pediatric patients we compared levels of cytokines and chemokines in the cerebrospinal fluid before and after three months on treatment to evaluate a possible anti-inflammatory effect. We analyzed 34 cytokines and chemokines in the cerebrospinal fluid of pediatric patients (n = 21) with refractory epilepsy by a multiplex assay. Beta-hydroxybutyric acid was measured in blood and cerebrospinal fluid. Seizure frequency in relation to diet treatment was assessed. For 9 different cytokines (CCL 7, CCL 21, CCL 22, CCL 25, CCL 27, IL-2, IL-10, CX3CL1 and MIF), a significant decrease ranging from 7 to 27% was seen after three months as compared to levels before the diet. In contrast, no cytokine displayed a significant increase during diet. A seizure reduction ≥ 50 % was seen in 15/21 patients (71 %) but no significant differences in cytokine decreases were found between responders and non-responders during treatment. A non-significant trend towards higher initial pre-treatment levels of cytokines was seen in responders, which were reduced following treatment. The levels of betahydroxybutyric acid were not related to seizure response. We conclude that while it is not possible to state a primary anti-inflammatory effect by dietary treatment from these data, an unequivocal immunological effect is seen and may be a part of the mechanism of ketogenic dietary treatment.

Post-encephalitic epilepsy in childhood: results from a prospective cohort study.

To investigate whether it is possible to predict outcome of post-encephalitic epilepsy based on findings during the acute phase of disease. Children (28 days to 17 years) diagnosed with acute encephalitis at Karolinska University Hospital between 2011 and 2016 were included in this study (n=89). They were examined clinically, with repeated electroencephalographic examinations and analysis of cerebrospinal fluid during the acute illness. Thereafter, patients were followed up to 24 months and evaluated for post-encephalitic epilepsy. Variables determined during the acute illness were used to predict the development of post-encephalitic epilepsy: electroencephalographic parameters, cerebrospinal fluid parameters, aetiology and clinical parameters. Fisher's exact test was used to estimate any predictors of epilepsy among the acutely measured parameters. The prevalence of post-encephalitic epilepsy was 9% (n=8) at 24 months. Of these, 3/8 responded to monotherapy with antiepileptic drugs and 5/8 required two or more and 3/8 were medically refractory at 24 months. Presence of acute seizures during admission, epileptic activity on electroencephalographic recordings and new-onset structural lesions demonstrated a significant association with development of post-encephalitic epilepsy (p<0.03) with an odds ratio greater than 5. Using the three above-mentioned parameters, we designed an algorithm to predict cohorts of patients with increased risk of developing post-encephalitic epilepsy. Moreover, patients who developed post-encephalitic epilepsy had a longer duration of hospital admission and longer care in intensive care units in comparison to those who did not. This study demonstrates that the risk of developing post-encephalitic epilepsy was mainly seen among patients with acute seizures, epileptic encephalographic activity in the acute setting or new-onset structural lesions. A simple algorithm could be used to predict the risk of post-encephalitic epilepsy.

The Kynurenine Pathway is Differentially Activated in Children with Lyme Disease and Tick-Borne Encephalitis.

In children, tick-borne encephalitis and neuroborreliosis are common infections affecting the central nervous system. As inflammatory pathways including cytokine expression are activated in these children and appear to be of importance for outcome, we hypothesized that induction of the kynurenine pathway may be part of the pathophysiological mechanism. Inflammatory biomarkers were analyzed in cerebrospinal fluid from 22 children with tick-borne encephalitis (TBE), 34 children with neuroborreliosis (NB) and 6 children with no central nervous system infection. Cerebrospinal fluid levels of kynurenine and kynurenic acid were increased in children with neuroborreliosis compared to the comparison group. A correlation was seen between expression of several cerebrospinal fluid cytokines and levels of kynurenine and kynurenic acid in children with neuroborreliosis but not in children with tick-borne encephalitis. These findings demonstrate a strong induction of the kynurenine pathway in children with neuroborreliosis which differs from that seen in children with tick-borne encephalitis. The importance of brain kynurenic acid (KYNA) in both immune modulation and neurotransmission raises the possibility that abnormal levels of the compound in neuroborreliosis might be of importance for the pathophysiology of the disease. Drugs targeting the enzymes of this pathway may open the venue for novel therapeutic interventions.

Integration of whole genome sequencing into a healthcare setting: high diagnostic rates across multiple clinical entities in 3219 rare disease patients.

BACKGROUND: We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. METHODS: Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout-a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. RESULTS: Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. CONCLUSIONS: Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

The Cerebrospinal Fluid Interleukin-6/Interleukin-10 Ratio Differentiates Pediatric Tick-borne Infections.

BACKGROUND: Borrelia burgdorferi and tick-borne encephalitis (TBE) virus are 2 types of tick-borne pathogens that can cause central nervous system infection. Routine diagnostics have so far included analysis of cerebrospinal fluid (CSF) cell numbers, CSF serology for Borrelia burgdorferi and serum serology for TBE virus. However, early diagnosis may be difficult based on antibody detection which takes time to analyze, and with the possibility of false negative results, thus delaying treatment. Cytokine analyses are becoming increasingly available in clinical routine care and may offer important information. METHODS: Fifteen cytokines and chemokines were measured in the CSF from the diagnostic lumbar puncture of 37 children with TBE, 34 children with neuroborreliosis and 19 children without evidence of central nervous system infection, using Luminex technology. RESULTS: Significantly higher levels of proinflammatory interleukin-6 were detected in the samples from TBE-infected children, when compared with neuroborreliosis or controls. In comparison, children with neuroborreliosis had significantly higher levels of interleukin-7, interleukin-8, interleukin-10, and interleukin-13 when compared with TBE infected or controls. Furthermore, the ratio between interleukin-6 and interleukin-10 was significantly different between the 2 types of tick-borne infections. CONCLUSIONS: The interleukin-6/interleukin-10 ratio can be used as a rapid diagnostic cue upon suspected tick-borne infection, enabling fast and correct treatment. Also, in serology-negative results, such information may strengthen a clinical suspicion.

Prospective Evaluation of Childhood Encephalitis: Predisposing Factors, Prevention and Outcome.

BACKGROUND: Acute encephalitis in childhood is a serious condition. The severity varies between studies, partly reflecting differences in study design where only severe cases from referral centers often are reported. The aim of this study was to prospectively study the clinical picture and etiology of acute encephalitis in childhood at a primary and tertiary pediatric hospital in Sweden. METHODS: All children with acute encephalitis were prospectively included from 2011 to 2016. Laboratory tests, investigations and follow-up were performed according to standardized study protocols. RESULTS: Eighty-nine children were included (46 female and 43 male) with a median age of 53 months. An etiology was established in 61/89. Tick-borne encephalitis virus, enterovirus and rotavirus dominated and 34% were caused by a virus preventable by vaccination. Immune-mediated encephalitis was seen in 7 children. An abnormal electroencephalography picture was seen in 77/86, pathologic findings on neuroimaging in 13/49, and 38/89 children had seizures. Sequelae were reported by 49%. A high prevalence of previous contact with child and adolescent psychiatry was seen and, although not statistically significant, the need for extra support at school before encephalitis and the presence of central nervous system disease in the family seemed to predispose for a longer hospital stay. CONCLUSION: Encephalitis is a condition with long-term consequences. Most children need admission to hospital, and many need surveillance in the intensive care unit. The etiology can be determined in a majority of cases, and 1/3 could have been prevented by vaccination. This study corroborates electroencephalography as a cornerstone in diagnosis.